The present invention relates to a method of promoting retinal neuron survival as well as preventing photoneuron degredation.
The retina is the light-sensitive portion of the eye. The retina contains the cones and rods (photoreceptors), the photosensitive cells. The rods contain rhodopsin, the rod photopigment, and the cones contain 3 distinct photopigments, which respond to light and transmit signals through successive neurons to ultimately trigger a neural discharge in the output cells of the retina, the ganglion cells. The signal is carried by the optic nerve to the visual cortex where it is registered as a visual stimulus.
In the center of the retina is the macula lutea, which is about 1/3 to 1/2 cm in diarneter. The macula provides detailed vision, particularly in the center (the fovea), because the cones are higher in density. Blood vessels, ganglion cells, inner nuclear layer and cells, and the plexiform layers are all displaced to one side (rather than resting above the ones), thereby allowing light a more direct path to the cones.
Under the retina is the choroid, a collection of blood vessels embedded within a fibrous tissue, and the pigmented epithelium (PE), which overlays the choroid layer. The choroidal blood vessels provide nutrition to the retina (particularly its visual cells). The choroid and PE are found at the posterior of the eye.
The retinal pigment epithelial (RPE) cells, which make up the PE, produce, store and transport a variety of factors that are responsible for the normal function and survival of photoreceptors. RPE are multifunctional cells that transport metabolites to the photoreceptors from their blood supply, the chorio capillaris of the eye. The RPE cells also function to recycle vitamin A as it moves between the photoreceptors and the RPE during light and dark adaptation. RPE cells also function as macrophages, phagocytizing the rhythmically-shed tips of the outer segments of rods and cones, which are produced in the normal course of cell physiology. Various ions, proteins and water move between the RPE cells and the interphotoreceptor space, and these molecules ultimately effect the metabolism and viability of the photoreceptors.
The Muller cell is the most prominent glial cell within the retina, and could also be important for maintaining the viability of visual cells. Muller cells traverse the entire retina in a radial direction from the ganglion cells to the external limiting membrane, a photoreceptor-photoreceptor and Muller cell-photoreceptor contact point. In addition to providing structural support, Muller cells regulate the control of ionic concentrations, degradation of neurotransmitter, removal of certain metabolites and may be a source of important factors that promote the normal differentiation of photoreceptor cells. Kljavin and Reh (1991), J. Neuroscience 11: 2985-2994. Although a search for defects in Muller cells has not specifically been examined, any disease or injury affecting their normal function most likely would have a dramatic influence on the health of rods and cones. Finally, the death of rod photoreceptors may influence the viability of cones. One common feature in degenerations involving mutations in rod specific genes (i.e., rhodopsin) is that cones also eventually die. The reason for the loss of cones has not been determined, although it has been suggested that dying rods may release endotoxins. Bird (1992), Opthal. Pediatric. Genet. 13: 57-66.
Diseases or injury to the retina can lead to blindness if retinal cells are injured or killed. The photoreceptor cells are particularly susceptible to injury since they are often the first cells to degenerate or suffer damage as a result of a traumatizing event or condition. Hereditary defects in specific photoreceptor genes, retinal detachment, circulatory disorders, overexposure to light, toxic effects to drugs and nutritional deficiencies are among the wide array of causes that can result in the death of photoreceptor cells. Developmental and hereditary diseases of the retina account for around 20 percent of all legal blindness in the United States. Report of the Retinal and Choroidal Panel: Vision Research--A National Plan 1983-1987, vol. 2, part I, summary page 2. For example, retinitis pigmentosa (RP), a genetic based progressive disease is characterized by incremental loss of peripheral vision and night blindness, which is due in large part to the loss of photoreceptor cells. RP is a group of hereditary diseases and presently afflicts about one in 3000 persons worldwide. Wong, F. (1995) Arch. Ophthalmol. 113: 1245-47. Total blindness is the usual outcome in more progressive stages of this disease. Macular degeneration, another major cause of blindness, is a complex group of disorders that affects the central or predominantly cone portion of the retina. Cones are primarily responsible for acute vision. Diabetic retinopathy, a frequent complication in individuals with diabetes mellitus, is estimated to be the fifth leading cause of new blindness. However, it is the second leading cause of blindness among individuals of 45-74 years of age. Moreover, these problems are only expected to get worse as the general population ages.
Photoreceptor degeneration may also occur as a result of overexposure to light, various environmental trauma or by any pathological condition characterized by death or injury of retinal neurons or photoreceptors.
Photoreceptor loss may also be influenced by cellular or extracellular retinal components. The primary example of extracellular stimulus is related to the close association between the pigment epithelium (PE) and the photoreceptor cells. As mentioned previously, the PE transports metabolites to and from the photoreceptors as well as removes discarded cellular material. Retinal detachment, which involves the separation of the neural retina from the PE leads to photoreceptor death. Furthermore, the degree of cell loss is dependent upon the duration of the separation. Gouras et al. (1991) IOVS 32: 3167-3174.
Additionally, diseases of the PE can lead to photoreceptor cell loss. The primary example of this is the Royal College of Surgeons (RCS) rat, which has an inherited retinal dystrophy due to a defect in the PE, resulting in photoreceptor cell death during the normal course of the animal's life. Mullen & LaVail (1976), Science 192: 799-801. In this animal, the PE are unable to phagocytize outersegment debris which accumulates between the photoreceptor cells and the PE, and as a result, provide a useful model system to study the role of trophic factors on the retina. A delay of photoreceptor death is obtained through the proximal placement of normal PE cells both in experimental chimeras, Mullen & LaVail, supra and by transplantation of PE from healthy animals. Li & Turner (1988), Exp. Eye Res. 47: 911-917; Sheedlo et al. (1992), Int. Rev. Cytol. 138: 1-49; Lavail et al. (1992), Exp. Eye Res. 55: 555-562; Lavail et al. (1992), PNAS 89: 11249-11253. In all of these experiments, the "rescue" extended beyond the boundaries of the normal PE cells, and suggests the presence of difussible trophic factor(s) produced by the PE cells.
Another useful animal model is the albino rat. In this animal, normal illumination levels of light, if continuous, can cause complete degeneration of photoreceptors. Results obtained using such rats as a model to identify survival enhancing factors appear to correlate well with data obtained using RCS rats. Moreover, different factors can be compared and complications can be assessed more quickly in the light damage model than can be assessed by testing factors in models which are based on the slowly evolving dystrophy of the RCS rat.
Using albino rats, it has been determined that a number of agents, when administered systemically (intraperitoneally) can be used to ameliorate retinal cell death or injury caused by exposure to light. In general, exposure to light generates oxygen free radicals and lipid peroxidation products. Accordingly, compounds that act as antioxidants or as scavengers of oxygen free radicals reduce photoreceptor degeneration. Agents such as ascorbate, Organisciak et al. (1985), Invest. Opthal. & Vis. Sci. 26: 1580-1588, flunarizine, Edward et al. (1991), Arch. Ophthalmol. 109: 554-562, and dimethylthiourea, Lam et al. (1990), Arch. Opthal. 108:1751-1757 have been used to ameliorate the damaging effects of constant light. There is no evidence, however, that these compounds will act to ameliorate other forms of photoreceptor degeneration and their administration can potentiate harmful side effects. Further, these studies are limited because they utilize systemic delivery, which does not provide an adequate means of assessing the effectiveness of a particular factor. As a result, it is nearly impossible to assess the amount of agent that actually reaches the retina. A large amount of agent must be injected to attain a sufficient concentration at the site of the retina. In addition, systemic toxic effects may result from the injection of certain agents.
Traditional approaches to treating the loss of vision due to photoreceptor cell death has taken at least two routes: (1) replacing the defective cells by physical transplantation; and (2) slowing, arresting or preventing the process of degeneration. The transplantation of healthy pigment epithelium cells into a degenerating retina or one which has defective epithelium cells can rescue photoreceptor cells from dying. Sheedlo et al. (1992), Int. Rev. Cytol. 138: 1-49); Lavail et al. (1992), Exp. Eye Res. 55: 555-562; and Lavail et al. (1992), PNAS 89: 11249-11253. PE transplants in humans have been attempted, but the results have been less than satisfactory. Peyman et al. (1991), Opthal. Surg. 22: 102-108. More promising, but as yet unproven is the transfer of embryonic retina containing mostly undifferentiated progenitor cells, which can differentiate in response to environmental cues into appropriate missing cell types. Cepko (1989), Ann. Rev. Neurosci. 12: 47-65. In conclusion, therapy via functional integration of transplanted retinal cells into a human host retinas remain unproven.
Other strategies have focused on "rescuing" or slowing the loss of visual cells. These techniques include corrective gene therapy, limiting the exposure to normal light during disease, vitamin A supplemented diets and the administration of growth factors to damaged or degenerating eyes. However, these treatment schemes have several limitations.
For example, gene therapy or the insertion of a replacement allele into the cells carrying the known mutation may prove problematic. Milam, Curr. Opin. Neurobiology 3: 797-804 (1993). Since rods and cones are somewhat inaccessible, it might be difficult to deliver replacement genes to them. Moreover, the use of retroviral vectors for insertion of replacement genes is limited to dividing cells, such as cultured PE, whereas post-mitotic neurons, e.g. photoreceptors, will require other viral vectors such as HSV (Herpes simplex virus) for effective delivery. Finally, gene replacement may not correct a disease where the mutant gene product is deleterious to the cell, but may be more useful for correcting defects due to the loss-of-function of a gene product, as is found in most recessive disorders.
Limiting light exposure, a low technology conventional approach to attenuating vision loss, typically using such approaches as eye-patches, dark goggles, etc. is impractical, since the practical effect of the treatment is the same as the disease itself: blindness and inability to detect light.
Vitamin A has been observed to halt the decline of retinal function by over 20% as administered over the course of 4-6 years in the progression of patients with retinitis pigmentosa (RP). E. L. Berson et al., Arch Ophthalmol. 111: 761-772 (1993). While this study did indicate a potential lengthening of years of useful vision, several criticisms of vitamin A therapy exist: (1) the mechanism by which vitamin A (and even vitamin E) alter the progression of RP is unknown; (2) it is not known whether or not patients with different genetic forms of RP will respond to vitamin A therapy; (3) it is not apparent whether or not quantifiable measurements of visual function (i.e., perimetry and visual acuity) revealed any significant benefit from vitamin A therapy; and (4) long term ingestion of vitamin A may have detrimental side effects in other organ systems.
A number of agents, when administered systemically (intraperitoneally) can be used to ameliorate retinal cell death or injury caused by exposure to light. In general, exposure to light generates oxygen free radicals and lipid peroxidation products. It has been suggested that genetically defective photoreceptors are abnormally sensitive to photooxidation from light levels as encountered normally in the environment. Hargrave, P A. & O'Brien, P J., Retinal Degenerations, Anderson R E et al. eds., Boca Raton, Fla., CRC Press, p. 517-528 (1991). Compounds that act as antioxidants or as scavengers of oxygen free radicals reduce photoreceptor degeneration. Anti-oxidants or calcium overload blockers (e.g. flunarizine) have been reported to prevent degeneration of normal photoreceptors after exposure to high light levels. Rosner et al., Arch. Ophthalmol 110: 857-861 (1992); Li et al. Exp. Eye Res. 56: 71-78 (1993). Additional success in reducing photoreceptor degeneration has been observed through administration of ascorbate (Organisciak et al., Invest. Ophthal. & Vis. Sci. 26: 1580-1588 (1985)), flunarizine (Edward et al., Arch. Ophthalmol. 109: 554-562 (1991)), and dimethylthiourea (Lam et al., Arch. Ophthal. 108: 1751-1757 (1990)). However, there is no evidence that administration of these compounds will reduce photoreceptor degeneration induced by other than intense light exposure. Moreover, there is great concern that their administration can generate potentially harmful side effects. As a result, the search continues for factors which can somehow protect photoreceptors or even promote their regeneration after light-induced damage.
A particular area of interest is the administration of growth factors. Growth factors have been found to participate in diverse roles such as neuronal differentiation, transmitter specificity, regulation of programmed cell death, and neurite growth in several regions of the central nervous system. However, only recently has their role been studied during retinal development and disease. An early study indicating that diffusible growth factors can rescue photoreceptor cells from dying was based on a chimeric rat constructed to contain both normal and RCS pigment epithelial cells. The animals were produced by fusing blastula from both normal and RCS rat embryos. Mullen and LaVail, supra. In the retina of these chimeras, photoreceptor cells adjacent to RCS PE showed degeneration, and those that were lying next to normal PE were healthy. However, photoreceptor cells that were lying just beyond the immediate contact site of normal PE also appeared healthy, suggesting that photoreceptor-PE contact was not needed, and that normal PE were secreting a putative survival promoting factor.
Among the best characterized growth factors in the retina are the acidic and basic fibroblast growth factors (aFGF and bFGF). FGF can be detected through immunohistochemical, biochemical or molecular approaches on a variety of retinal cells including PE, photoreceptor cells and the interphotoreceptor cell matrix (IPM), and a collection of extracellular matrix molecules surrounding photoreceptor cells. Jacquemin et al. (1990) Neurosci. Lett. 116: 23-28; Caruelle et al. (1989) J. Cell Biol. 39: 117-128; Hageman et al. (1991) PNAS 88: 6706-6710; Connolly et al. (1991) IOVS 32 (suppl.): 754Intravitreal injection of basic fibroblast growth factor (bFGF) in the RCS rat or rats with light damaged retina prevents photoreceptor cell degeneration for several month, even as outersegment debris accumulates. Faktorovich et al. (1990), Nature 347: 83-86. Similar results have been seen when bFGF is injected into the subretinal space, the area between the photoreceptors and the PE. However, even sham operations, or injections of phosphate buffered saline (PBS) in both the RCS rat and light damaged retina can delay photoreceptor cell death. However, the rescue effect is small and localized to the needle track, and differs quantitatively from the effect obtained from bFGF. Faktrorovich et al., supra; Silverman and Hughes (1990), Curr. Eye Res. 9: 183-191; Sheedlo H. J. et al., Int. Rev. Cyto. 138: 1-49 (1992). In these experiments it is likely that various growth factors derived from damaged retinal tissues or macrophages present in the damaged area were locally released. Sheedlo et al., supra.; Silverman and Hughes, supra. Macrophages themselves are known to produce many different growth factors or cytokines, some of which could have photoreceptor survival activity. Rappolee and Werb, Curr. Top. Microbiol. Immunol. 181: 87-140 (1992).
Various agents disclosed to have survival-enhancing and/or growth activity on retinal neurons are described in certain issued patents and pending patent applications. These include Transforming Growth Factor-.beta. (TGF-.beta.) (WO 94/01124), brain derived neurotrophic factors (BDNF) (U.S. Pat. No. 5,180,820) (U.S. Pat. No. 5,438,121) and (WO 91/03568), neurotrophin-4 (NT-4) (WO 93/25684), and insulin-like growth factors (IGF) (WO 93108826).
Other experiments have shown that intravitreal injections of human subretinal fluid as well as other growth factors can rescue dying photoreceptor cells. For example, one recent study tested eight different factors injected into the retina of rats exposed to constant high intensity light, all showing the ability to delay the degeneration of photoreceptor cells. These include FGF (both acidic and basic forms), brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and interleukin 1 (IL-1). Neurotrophin 3 (NT 3), insulin-like growth factor II (IGF-II), Transforming Growth Factor beta (TGF-.beta.) and the tumor necrosis factors alpha and beta (TNF-.alpha., TNF-.beta.) also showed survival activity, but to a much lesser degree than the other factors. NGF has been reported to reduce the incidence of apoptosis in diabetic rats in addition to minimizing pericyte loss and acellular occluded capillaries, conditions associated with diabetic retinopathy. Hammes, H P et al., Molecular Med. 1(5): 527-534 (1995). However, while it does appear that growth factors can enhance survival of photoreceptors, some of these factors may promote detrimental side effects. For example, injections of bFGF results in an increased incidence of macrophages and cataracts. In addition, bFGF is mitogenic for PE, Muller cells and retinal vascular cells. Faktorovich et al., supra.; La Vail et al., supra. As a result, suitable growth factors which will not only promote the survival of photoreceptor cells, but lack undesired side effects have yet to be discovered.
FGF-5 is a member of the fibroblast growth factors (FGF's) which are family of potent mitogens for both normal diploid fibroblasts and established cell lines, Gospodarowicz, D. et al. (1984) Proc. Nat'l. Acad. Sci. USA 81:6963FGF-5 was originally identified as a transforming gene by the NIH-3T3 focus formation assay using DNA derived from human tumors. This protein was originally identified as a 267 amino acid residue polypeptide with a putative 22 amino acid residue signal peptide. The FGF family comprises acidic FGF, basic FGF, INT-2 (FGF-3), K-FGF/HST (FGF-4), FGF-5, FGF-6, KGF (FGF-7), AIGF (FGF-8), FGF-9, FGF-10, etc. Recently, a new member of this family, designated FGF-16 has been isolated (Miyake et al., Biochem. Biophys. Res. Commun. 243(1): 148-152 (1998). FGFs typically have two conserved cysteine residues and share 30-50% sequence homology at the amino acid level. These factors are mitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-derived cells, including granulosa cells, adrenal cortical cells, chondrocytes, myoblasts, corneal and vascular endothelial cells (bovine or human), vascular smooth muscle cells, and lens epithelial cells. The mitogenicity of FGF-5 is particularly described by 3H-thymidine incorporation in quiescent NR6R-3T3 fibroblasts in Thomas, K., Methods in Enzymology 147: 120-135 (1987).
FGF-5 was first disclosed as the gene product of an oncogene called ORF-2, Goldfarb, M. et al., WO 88/09378; Zhan X., et al. (1987) Oncogene 1: 369-376; Zhan et al. (1988) Mol. Cell. Biol. 8: 3487-3495The protein was originally called FGF-3, owing to its similarity to the previous known a-FGF and b-FGF. However, by the time that the sequence of this molecule had been made publicly available, two additional FGF-related polypeptides, two additional FGF-related polypeptides, INT-2 and HST/K-FGF, had already been described, Zhan et al., 1988, supra. As a result, FGF-3 was redesignated FGF-5. Subsequently, it was found that the sequence described in Zhan et al. is incorrect, and the correct sequence appears in Haub et al., Proc. Natl. Acad. Sci. USA 87: 8022-8026 (1990).
FGF-5 has been described to promote the survival and growth of motor neuron cells, and was proposed for the treatment of diseases characterized by the dysfunction of motor neurons (WO 94/20125). FGF-5 was also shown to have activity in the promotion, survival and differentiation of cholinergic septal and serotonergic neurons (WO 95/15176; Lindholm et al., Eur. J. Neurosci. 6: 244-252 (1994)). Recombinant FGF-5 (R&D systems) is also known to be mitogenic in Balb/3T3 fibroblasts and bovine heart endothelial cells. R & D Systems data sheet, FGF-5, Cat. No. 237-F5/CF. FGF-5 has also been found to be expressed in the PE, Bost et al., Exp. Eye Res. 55: 727-734 (1992), as well as ganglion cells and photoreceptors, Reh et al., Ciba Found. Symp. 196: 120-131 (1996).
However, FGF-5 has not been previously known as a potential survival promoting agent for photoreceptor cells. This is likely due to at least two reasons: (1) Various other known FGFs are mitogenic for retinal cells, especially basic FGF; (2) FGF-5 is mitogenic in fibroblast, endothelial and motor neuronal cells. As a result, its homology to other retinal mitogenic agents as well as the mitogenic character upon non-retinal cells would lead one of ordinary skill in the art to expect FGF-5 to be mitogenic to retinal cells as well.
Surprisingly, Applicants have discovered that FGF-5 prevents the death of photoreceptor cells without any significant mitogenic effect upon photoreceptor cells. Thus, it appears to be an ideally suited candidate for a photoreceptor and/or retinal neuron survival agent.